Abstract: This renewal continues our optimization of myeloid-derived suppressor cell (MDSC) infusion for creating transplant tolerance to prevent graft-vs-host disease (GVHD) in the clinic. Classical immunosuppressants do not inhibit the initial, innate response critical for tissue inflammation. Myeloid-derived suppressor cells (MDSCs) are innate immune suppressors, functioning by fundamentally different mechanisms, which we hypothesize can suppress the early innate immune cell activation and may participate in tissue injury repair. We showed that highly suppressive murine MDSCs could be generated in short-term culture of normal bone marrow and that monocytic MDSCs (M-MDSCs) were the most potent subset in suppressing GVHD. Because MDSCs can be antigen-independent suppressors, off-the-shelf MDSC preparations for clinical trial(s) are practical. A limitation in translation has been the inherent batch-to-batch variability of primary donor-derived MDSCs. To solve batch variability, we will use transgene-free, clone-based induced pluripotent stem cells (IPSCs) that can be differentiated into suppressive M-MDSCs in 19 days, expandable by 1000-fold, and have phenotypic and functional characteristics of healthy donor peripheral blood MDSCs. This differentiation system was developed by our grant collaborators at Fate Therapeutics who have worked with us/our program to export protocols and a preselected IPSC clone to optimize GMP methods for a planned 2018 IPSC NK cancer trial. Unexpectedly, the greatest remaining impediment is MDSC propensity to terminally differentiate into mature non-suppressive antigen-presenting cells in the inflammatory GVHD milieu, also seen in inflammasome activated human primary MDSCs. With our grant collaborators, we systematically identified key molecular targets to overcome these limitations. Our scientific premise is gene engineering of indefinitely self-renewing IPSCs will subvert inherent MDSC instability in an inflammatory milieu and prolong their in vivo lifespan, creating a single, uniform well-characterized off-the-shelf M-MDSC batch meeting pre- release criteria. We will test the hypotheses that: Aim 1. Human IPSC or iCD34 gene engineering will provide off-the-shelf, third-party M-MDSCs with optimized antigen-independent GVHD suppression by CRISPR/Cas9 gene disruption of pathways controlling inflammasome activation (NLRP3, shared ASC subunit; ATP receptors P2x7R, P2Y14), inflammatory monocyte differentiation (batf3), and monocyte to macrophage conversion (NFKB1 p50). To prolong in vivo lifespan, we will test c-FLIP overexpression in M-MDSCs. Robust in vitro and in vivo testing in xenogenic GVHD mice will assess key suppression mechanisms and rank potency. Aim 2. Human anti-AML specific T cell killing of acute myeloid leukemia (AML) blasts will be unimpaired using gene engineered M-MDSCs that preferentially home to inflammation sites. By pinpointing key pathways for M-MDSC viability and suppression and harnessing IPSC technology, our expert team will gain mechanistic insights into controlling GVHD and preserving anti-AML specific T cell responses, providing a platform for GVHD trials.